The ecological consequences and evolution of retron-mediated suicide as a way to protect Escherichia coli from being killed by phage

Abstract

Retrons were described in 1984 as DNA sequences that code for a reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). It would not be until 2020 that a function was shown for retrons, when compelling evidence was presented that retrons activate an abortive infection pathway in response to bacteriophage (phage) infection. When infected with the virulent mutant of the phage lambda, λVIR, and to a lesser extent, other phages, a retron designated Ec48 is activated, the Escherichia coli bearing this retron element dies, and the infecting phage is lost. With the aid of a mathematical model, we explore the a priori conditions under which retrons will protect bacterial populations from predation by phage and the conditions under which retron-bearing bacteria will evolve in populations without this element. Using isogenic E. coli with and without Ec48 and λVIR, we estimated the parameters of our model and tested the hypotheses generated from our analysis of its properties. Our models and experiments demonstrate that cells expressing a retron-mediated abortive infection system can protect bacterial populations. Our results demonstrate that retron bearing bacteria only have a competitive advantage under a limited set of conditions.

Fig 1. Diagram of a model of the population and evolutionary dynamics of lytic phage and bacteria with and without a retron-mediated abortive infection mechanism.

There is a single population of lytic phage, P; a phage-sensitive retron-encoding (retron⁺) population, E; an envelope resistant retron⁺ population, E_r; a phage-sensitive (retron^-) population, N; and an envelope resistant retron^- population, N_r. The phage adsorbs to the N and E bacteria with rate constants, δ_n and δ_e (ml·cells/hour), respectively. The phage replicates on the N population with each infection producing β_n phage particles, the burst size. A fraction q (0 ≤ q ≤1) of the phages that adsorb to E population are lost and thus do not replicate. The remaining (1—q) of infections of E produce β_e phage particles. At rates μ_nr and μ_er per cell per hour, the bacteria transition from their respective phage sensitive to phage resistant states, and at rates μ_rn and μ_re they transition from the resistant to their respective phage sensitive states.

Fig 2. Conditions under which retrons protect monoclonal bacterial populations from phage infection.

Computer simulations results without envelope resistance and experimental results. The computer simulations model dynamics in the liquid environment. Shown are the densities of a retron⁺ bacterial population in the absence (blue) and presence (orange) of phage (red) and a retron^- bacterial population in the absence (green) and presence (purple) of phage at 0 (Initial) and 24 hours (Final). The parameters used for the simulations were: k = 1, e = 5x10^-7 ug/cell, v_e = v_n = 2.0 h^-1, δ_e = δ_n = 2x10^-7 h^-1cell^-1, β_e = β_n = 60 phages/cell, μ_nr = μ_rn = μ_er = μ_re = 0. A, B- Computer simulation results with a completely effective (A, q = 1.00) and incompletely effective (B, q = 0.95) abortive infection system. C, D, E- Protection experiments in liquid (C), soft agar (D), and with colonies growing on a surface (E). Plotted are means and standard deviation of three biological replicas.

Fig 3. Conditions for the invasion of a retron⁺ population into a community dominated by a retron^- population.

Computer simulations in the absence of envelope resistance, and experimental results. Bacteria and phage densities at time 0 (Initial) and 24 hours (Final). Left side: retron⁺ (blue) and a retron^- bacterial population (green) co-cultured in the absence of phage. Right side: retron⁺ (orange) and a retron^- bacterial population (purple) co-cultured in the presence of phage (red). The parameters used for the simulations were: k = 1, e = 5x10^-7 ug/cell, v_e = v_n = 2.0 h^-1, δ_e = δ_n = 2x10^-7 h^-1cell^-1, β_e = β_n = 60 phages/cell, μ_nr = μ_rn = μ_er = μ_re = 0, q = 1.00. A- Computer simulation results for an invasion condition with a completely effective abortive infection system. B, C, D- Invasion experiments in liquid (B), soft agar (C), and with colonies growing on a surface (D). Shown are means and standard deviation of three independent experiments with biological replicas.

Fig 4. Computer simulations of retron population dynamics with envelope resistance.

The simulation conditions are similar to those in Fig 3A but allow for the generation of resistance, μ_er = μ_re = μ_nr = μ_rn = 10⁻⁵ per cell per hour. Densities of bacteria and phage at time 0 (Initial) and 24 hours (Final) for two invasion conditions. Left side: retron⁺ (blue) and retron^- (green) bacteria in the absence of phage. Right side: retron⁺ (orange) and retron^- bacteria (purple) in the presence of phage (red). The densities of phage-resistant mutant bacteria are noted by bars with white hashing next to the bar of the sensitive population. A- Simulations with a completely effective abortive infection system (q = 1.00). B- Simulations with a less-than completely effective abortive infection system (q = 0.95).